Journal: Structure / Year: 2010 Title: Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR. Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John ...Authors: Goragot Wisedchaisri / David M Dranow / Thomas J Lie / Jeffrey B Bonanno / Yury Patskovsky / Sinem A Ozyurt / J Michael Sauder / Steven C Almo / Stephen R Wasserman / Stephen K Burley / John A Leigh / Tamir Gonen / Abstract: Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. ...Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
Resolution: 2.5→2.59 Å / Redundancy: 3.8 % / Rmerge(I) obs: 0.59 / Mean I/σ(I) obs: 1 / Rsym value: 0.59 / % possible all: 98.8
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Processing
Software
Name
Version
Classification
NB
REFMAC
refinement
PDB_EXTRACT
3.1
dataextraction
MAR345
CCD
datacollection
HKL-2000
datareduction
HKL-2000
datascaling
SHELXCD
phasing
SHELXE
modelbuilding
RESOLVE
phasing
Refinement
Method to determine structure: SAD / Resolution: 2.5→33.15 Å / Cor.coef. Fo:Fc: 0.93 / Cor.coef. Fo:Fc free: 0.91 / WRfactor Rfree: 0.259 / WRfactor Rwork: 0.233 / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.909 / SU B: 27.562 / SU ML: 0.278 / SU R Cruickshank DPI: 1.955 / SU Rfree: 0.348 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R Free: 0.348 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.277
785
5.1 %
RANDOM
Rwork
0.246
-
-
-
obs
0.247
15303
99.3 %
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all
-
15411
-
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK